β-Lactam Resistance Genes: Characterization, Epidemiology, and First Detection of blaCTX-M-1 and blaCTX-M-14 in Salmonella spp. Isolated from Poultry in Brazil-Brazil Ministry of Agriculture's Pathogen Reduction Program.

Salmonella spp. are widespread in nature; however, human infections occur mainly through ingestion of contaminated food, specially poultry and eggs. In Brazil, the Ministry of Agriculture (MAPA) oversees food production in general, with the goal of preventing transmission of pathogens through the food chain.

The road to the discovery of dendritic cells, a tribute to Ralph Steinman.

While it was known by the 1960s that lymphocytes mediated adaptive immunity, it was unknown how antigens stimulated lymphocytes. Between 1967 and 1973, we reported that a rare cell type in murine spleen cells took up antigen and were obligatory for T cell dependent and independent antibody responses. We referred to them as A cells or the third cell type.

Prevention of renal allograft rejection without immune suppression: a model to revisit.

Spectacular success in preventing renal allograft rejection in rats was obtained over 40 yr ago using only the reactants of the response: donor-type antigen and homologous antiserum directed against donor-type antigen. Tolerance was antigen specific and sustained by persistent antigen of the graft. The model has never been tested rigorously in a large species, though the rationale for why the procedures should work applies across species including humans.

Production of TH1 and TH2 cell lines and clones.

This unit describes protocols for the generation of polyclonal T(H)1 and T(H)2 cell lines from naive CD4(+) T cells as well as for generation of antigen-specific cell lines from TCR-transgenic mice and antigen-specific T cell clones from primed mice. Also described are methods for the preparation and maintenance of alloreactive murine helper T (T(H)) lymphocyte and cytotoxic T lymphocyte (CTL) clones using the limiting dilution technique, as well as derivation of T(H) clones reactive with soluble protein antigens, including a method for the selection of either T(H)1 or T(H)2 lymphocyte subsets. These two subsets of T(H) cells exhibit helper function in different ways and can be distinguished by the patterns of cytokines they synthesize.

Diagnosis and treatment of mycoplasma-contaminated cell cultures.

Mycoplasma contamination is a serious and frequent problem in the culture laboratory. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. This appendix describes how to test for mycoplasma contamination, and also presents methods for antibiotic treatment of infected cultures.

B7.1 is a quantitatively stronger costimulus than B7.2 in the activation of naive CD8+ TCR-transgenic T cells.

Using a TCR transgenic mouse bred onto a recombinase-activating gene-2-deficient background, we have examined the influence of B7.1 and B7.2 on activation of naive, CD8+ T cells in vitro.

IL-4 enhances long-term survival of CD28-deficient T cells.

CD28 signaling is critical for IL-2 production by established Th1 clones, but CD28 does not appear to play a role in the activation of established Th2 clones. To determine the role of CD28 in the generation of polarized T cells, clones were derived using cells from CD28-deficient (CD28-/- mice, which had been bred with mice that express the DO11.10 transgene, a CD4+ TCR-alphabeta receptor that recognizes OVA peptide 323-339 bound to I-Ad.

CD28 costimulation promotes the production of Th2 cytokines.

CD28 ligation augments TCR-mediated proliferation, IL-2 production, and T cell survival. However, the role of CD28 costimulation in T cell differentiation remains controversial. To address this issue, CD28+ and CD28-deficient TCR alphabeta transgenic (Tg) mice were used to examine cytokine production by T cells following antigenic stimulation.

Control of T lymphocyte signal transduction through clonal anergy.

Stimulation of interleukin-2 producing T lymphocytes via the T cell receptor (TCR) complex in the absence of other costimulatory factor results paradoxically not in activation but in an unresponsive state termed clonal anergy. T cell anergy appears to be a mechanism by which potentially autoreactive T lymphocytes are inactivated in the periphery, thus maintaining tolerance to self antigens. The breakdown of such tolerance may result in autoimmune diseases.

TCR-gamma delta cells in CD3 zeta-deficient mice contain Fc epsilon RI gamma in the receptor complex but are specifically unresponsive to antigen.

Unlike TCR-alpha beta cells, TCR-gamma delta cells express a distinct member of the zeta family, the gamma-chain of Fc epsilon RI (Fc epsilon RI gamma) within the TCR complex. To study the role of the Fc epsilon RI gamma-chain in TCR-gamma delta cells, a TCR-gamma delta transgenic mouse (G8) has been crossed with CD3 zeta-chain-deficient mice (G8.zeta-/-).

Blocked Ras activation in anergic CD4+ T cells.

T cell anergy is a state of functional unresponsiveness characterized by the inability to produce interleukin-2 (IL-2) upon T cell receptor stimulation. The mitogen-activated protein kinases ERK-1 and ERK-2 and the guanosine triphosphate-binding protein p21ras were found to remain unactivated upon stimulation of anergic murine T helper cell 1 clones. The inability to activate the Ras pathway did not result from a defect in association among Shc, Grb-2, and murine Son of Sevenless, nor from a defect in their tyrosine phosphorylation.

Induction of lytic pathways in T cell clones derived from wild-type or protein tyrosine kinase Fyn mutant mice.

The OVA-reactive CD4+ Th1 clones and alloreactive CD8+ clones derived from wild-type or fyn-/- mice serve as model systems which have allowed us to investigate several aspects of the molecular events associated with T cell-mediated cytotoxicity, including 1) the differential utilization of two distinct cytolytic pathways by CD4+ Th1 clones and CD8+ CTL, 2) a comparison of the pathways of lysis induced by stimulation of the TCR or by alternative stimuli, 3) the requirement of Fyn for derivation of antigen-specific T-cell clones having properties of CD4+ Th1 and CD8+ CTL cells 4) the differential requirement of Fyn in the induction of responses by TCR and the alternative stimuli. Stimulation through the TCR, either by APC bearing relevant antigen or by immobilized anti-CD3 mAb, resulted in comparable levels of target cell lysis by clones from both wild-type and fyn-/- mice. These clones also utilize the Fas pathway to lyse target cells.

Induction of the increased Fyn kinase activity in anergic T helper type 1 clones requires calcium and protein synthesis and is sensitive to cyclosporin A.

Several alterations in T cell receptor-associated signal transduction have been observed following induction of anergy of T helper type 1 (Th1) clones, including a modified intracellular free calcium ([Ca2+]i) response and increased kinase activity associated with the protein tyrosine kinase p59fyn. In the current study, we demonstrate that, although the kinetics of acquisition of both of these signaling alterations correlated with the generation of anergy, a normal calcium response returned within 48 h after removal from the anergizing stimulus, whereas the increased p59fyn activity persisted and the cells remained hyporesponsive. Generation of both the anergic state and the increased p59fyn activity was prevented in the presence of calcium-free medium, cycloheximide (CHX), or cyclosporin A (CsA), and could be mimicked by the calcium ionophore ionomycin.

Differential requirement for protein tyrosine kinase Fyn in the functional activation of antigen-specific T lymphocyte clones through the TCR or Thy-1.

The protein tyrosine kinase Fyn has been shown to be involved in signal transduction through the TCR and the glycosyl-phosphatidylinositol-linked surface molecule Thy-1 expressed on T cells. In this study, we examine the requirement for Fyn expression in signaling through the TCR or Thy-1 using a panel of Ag-specific T cell clones derived from fyn-/- mutant mice. These clones do not express normal Fyn protein, as measured by immune-complex kinase reaction using anti-Fyn Ab.

IL-4-producing CD8+ T cell clones can provide B cell help.

Interactions between CD4+ T cells and B cells are mediated by both soluble factors and cell surface molecules. The Ag-independent interaction between the CD40 ligand, expressed on activated T cells, and its CD40 receptor, expressed on B cells, enhances B cell proliferation in response to IL-4 stimulation. The expression of the CD40 ligand is induced on CD4+ T cells by stimulation with Ag-pulsed APC or mitogens.

Regulation of T lymphocyte subsets.

Patterns of cytokine secretion and functional differences distinguish T lymphocyte subsets. T lymphocyte subsets are also regulated differentially. Most established CD8+ lymphocyte clones secrete gamma-interferon (IFN-gamma) but not interleukin 2 (IL-2) or IL-4.

IL-4 treatment of small splenic B cells induces costimulatory molecules B7-1 and B7-2.

IL-4 has been shown to be involved in the early stages of B cell maturation. Changes induced by IL-4 include cell enlargement, increased viability, and increased MHC class II expression. However IL-4 alone does not induce B cell activation as defined by proliferation, lymphokine production, or Ig class switching.

Unique antigen recognition by a herpesvirus-specific TCR-gamma delta cell.

TCR-gamma delta cells, a T cell subset present in the epithelial and lymphoid tissues, have been implicated in viral and bacterial infections. We have identified a TCR-gamma delta clone (TgI4.4) that, unlike TCR-alpha beta cells, recognizes a herpes simplex virus type 1 transmembrane glycoprotein, gI, in an MHC class I- and class II-independent fashion.

"Anergy" of TH0 helper T lymphocytes induces downregulation of TH1 characteristics and a transition to a TH2-like phenotype.

Mature CD4+ helper T lymphocytes have been categorized into two major functional phenotypes, TH1 and TH2, which produce distinct arrays of lymphokines and which are thought to arise from a pluripotential precursor cell termed TH0. Clonal anergy can be induced in TH1 clones by stimulating via the T cell receptor (TCR) complex in the absence of a costimulator molecule; however, anergy has been difficult to demonstrate in TH2 clones. We show here that treatment of cloned TH0 lines with anergizing stimuli results in the selective loss of TH1 characteristics and retention of a TH2 phenotype.

Anergic T-lymphocyte clones have altered inositol phosphate, calcium, and tyrosine kinase signaling pathways.

Full activation of TH1 helper T lymphocytes requires ligation of the specific T-cell antigen receptor (TCR) and a second signal provided by costimulator molecule(s) expressed on particular antigen-presenting cells. Stimulation via the TCR complex alone generates a subsequent unresponsive state characterized by an inability to produce interleukin 2. We report here that such anergic cells exhibit multiple alterations in TCR-associated signaling.

Requirements for activation of CD8+ murine T cells. I. Development of cytolytic activity.

Cytolytic effector function fails to develop if proliferation of allospecific cytolytic T lymphocyte precursors is inhibited, but the requirements for generation of cytolytic activity have not been fully defined. In contrast, the cytolytic effector function of cytolytic T lymphocyte clones does not change during the cell cycle, and the level of cytolytic activity is independent of cellular proliferation. The requirement for proliferation by primary responding populations may reflect the need for clonal expansion of a few inherently cytolytic effector cells in order to reach a threshold number which can readily be detected in conventional cytolytic assays.